Bottom Line:
The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies.Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection.These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem.

fig03: Analysis of the KCNQ5 protein and mRNA levels in the CN after cochlear ablation. A: Western blots show levels of KCNQ5 and calretinin in the CN extracts from unmanipulated controls and rats 40 days after bilateral cochlear ablation (40d). Detection of GAPDH was included as a loading control. No significant differences were found in KCNQ5 protein density 40 days after cochlear removal compared with the controls (P70). Calretinin expression levels were significantly reduced. Values are expressed as the relative fold change percentage compared with the controls. B: KCNQ5 mRNA expression levels in the CN of the unmanipulated control rats and the rats in which the cochleae had been removed. Quantitative real-time RT-PCR showed no significant differences in the relative levels of KCNQ5 mRNA between control rats and rats 3 hr (3h) and 40 days (40d) after cochlear ablation (P > 0.05). Bars represent mean ± SEM of nine independent determinations. ***P < 0.001.

Mentions:
Regulation of KCNQ5 levels 40 days after cochlear removal was also analyzed by Western blotting (Fig. 3A). The densitometry analysis revealed that the overall KCNQ5 protein density in the CN 40 days after cochlear removal was statistically similar to that of the controls (86.695% ± 25.15% compared with the control group; P = 0.5839, unpaired t-test with Welch's correction). Samples included tissue trace amounts from the dorsal cochlear nucleus (DCN). These results support the conclusion that de novo KCNQ5 immunolabeling in neuronal cell bodies of the AVCN 40 days after cochlear removal reflects protein accumulation at levels comparable to those found in normal animals in endbulbs of Held, which degenerated as a result of cochlear removal. As an internal positive control for Western blotting, we also tested the calretinin levels, which had dropped dramatically (about 60% compared with the control group; P < 0.001, unpaired t-test with Welch's correction) 40 days after bilateral cochlear ablation (Fig. 3A). This corroborated, at the same time, loss of the nerve fibers seen with immunocytochemistry and Fluoro-Jade labeling.

fig03: Analysis of the KCNQ5 protein and mRNA levels in the CN after cochlear ablation. A: Western blots show levels of KCNQ5 and calretinin in the CN extracts from unmanipulated controls and rats 40 days after bilateral cochlear ablation (40d). Detection of GAPDH was included as a loading control. No significant differences were found in KCNQ5 protein density 40 days after cochlear removal compared with the controls (P70). Calretinin expression levels were significantly reduced. Values are expressed as the relative fold change percentage compared with the controls. B: KCNQ5 mRNA expression levels in the CN of the unmanipulated control rats and the rats in which the cochleae had been removed. Quantitative real-time RT-PCR showed no significant differences in the relative levels of KCNQ5 mRNA between control rats and rats 3 hr (3h) and 40 days (40d) after cochlear ablation (P > 0.05). Bars represent mean ± SEM of nine independent determinations. ***P < 0.001.

Mentions:
Regulation of KCNQ5 levels 40 days after cochlear removal was also analyzed by Western blotting (Fig. 3A). The densitometry analysis revealed that the overall KCNQ5 protein density in the CN 40 days after cochlear removal was statistically similar to that of the controls (86.695% ± 25.15% compared with the control group; P = 0.5839, unpaired t-test with Welch's correction). Samples included tissue trace amounts from the dorsal cochlear nucleus (DCN). These results support the conclusion that de novo KCNQ5 immunolabeling in neuronal cell bodies of the AVCN 40 days after cochlear removal reflects protein accumulation at levels comparable to those found in normal animals in endbulbs of Held, which degenerated as a result of cochlear removal. As an internal positive control for Western blotting, we also tested the calretinin levels, which had dropped dramatically (about 60% compared with the control group; P < 0.001, unpaired t-test with Welch's correction) 40 days after bilateral cochlear ablation (Fig. 3A). This corroborated, at the same time, loss of the nerve fibers seen with immunocytochemistry and Fluoro-Jade labeling.

Bottom Line:
The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies.Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection.These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem.